METHODS AND PLATFORMS 



Rubisco abundance will be determined by titration against the synthetic, noncompetitive 

 inhibitor carboxyarabinitol bisphosphate (CABP) or by labelling with radioactive CABP or by 

 immunological staining. The maximum activity and activation state will be measured in vitro 

 using 14 C bicarbonate as a tracer. In vivo activity will be obtained from photosynthesis-light 

 response curves determined from 14 C uptake using a photosynthetron. Photosynthetic pigments 

 will be determined by HPLC. 



All research will be ship-based, using conventional CTD sampling. Water volumes of 8-10 

 L will be required for PI curves, Rubisco and HPLC pigment determinations. Continuous access 

 to surface water perhaps through a non-toxic on-line system would allow intensive temporal 

 sampling appropriate to examining short term (including diel) variability. 



Requirements include bench space and a dedicated fume hood in which radioisotope work 

 can be performed. 



STRENGTHS AND LIMITATIONS OF PROPOSED RESEARCH 



Rubisco is one of the most abundant enzymes in phytoplankton. It is the enzyme which 

 catalyzes C0 2 assimilation and as such is a key point in the regulation of the rate of 

 photosynthesis. The principle strength is the contribution of this research to a mechanistic 

 understanding of variations in Pm and turnover time for photosynthetic electron. These variations 

 occur in response to time of day and physiological history with respect to light and nutrients. 



Limitations: The technique is time consuming and thus the number of samples that can be 

 processed is limited. There are likely to be interspecific variations in extraction efficiency, 

 activation state and maximum catalytic activity (in fact, one goal to the research is to assess the 

 extent of these variations.) 



STATUS OF RESEARCH 



This research is at the proof of concept stage. To date, laboratory results with the diatom 

 Thalassiosira weissflogii and Phaeodactylum tricornutum have demonstrated: (1) The activation 

 state of Rubisco is variable and this variation is correlated with the diel variability of Pm. (2) 

 The activation state changes in response to light-dark transitions, deactivating in the dark to 15% 

 of the maximum level observed in the light. (3) The rates of activation and deactivation of 

 Rubisco in diatoms have very similar time constants to those observed in higher plants. (4) The 

 kinetics of activation and deactivation of Rubisco have the same time constants as in vivo 14 C 

 uptake. We are currently attempting to optimize extraction procedures and modify existing 

 protocols to obtain quantitative measurements of Rubisco concentration in diatoms. 



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